Test method using cells and test kit therefor

ABSTRACT

The present invention provides a method for performing a biological test under conditions in which an artificially prepared cell pattern with initial position coordinates that can be determined is three-dimensionally cultured within a gelled matrix. The present invention relates to a biological test method that comprises testing a biological indicator with reference to at least one selected from the group consisting of cell proliferation, cell movement, and cell differentiation in a cell pattern substantially embedded in gel. The present invention also relates to a kit for the biological test method.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a biological test method using cells,which is used in the fields involving pharmaceuticals, medical care,foods, medicine, pharmacy, biology, and the like. The present inventionparticularly relates to a biological test method using three-dimensionalculture considered to enable obtainment of information that is moreuseful than that obtained via conventional two-dimensional culture. Thebiological test method is technology that is worthwhile as analternative to animal testing.

2. Background Art

In recent years, lower investment efficiency in research and developmenthas become an issue in the pharmaceutical industry. Hence, biologicaltest methods using three-dimensional culture methods are attractingattention as methods useful for reducing the high costs of animalexperimentation. Such biological test methods using three-dimensionalculture methods are expected to be useful for obtainment of basic datathat enable in vivo prediction of the absorption, distribution,metabolism, excretion, and toxicity of new drugs in particular. Thistechnology will influence not only the pharmaceutical industry, but alsoon treatment and diagnosis in the fields of medical care, food safetytests, and the like. Also, rapidly developed from research using aconventional two-dimensional culture method is research using acombination of cellular imaging technology and a three-dimensionalculture method. Practical use of screening technology that constitutes acombination of cellular imaging technology and a three-dimensionalculture method has already been initiated.

A conventional three-dimensional culture method will be described below.The most simple three-dimensional culture method involves culturingcells dispersed in a gel matrix such as collagen or agarose gel in anappropriate container. Furthermore, a method that involves forming a gellayer on the bottom of a culture container, seeding cells on the bottom,and then culturing the cells is also regarded as a three-dimensionalculture method when the cells migrate or infiltrate into the gel.Non-patent Document 1 discloses a method, namely a sandwich culturemethod that involves forming a gel layer on the bottom of a culturecontainer, seeding cells on the gel layer, culturing the cells, removingmedium after a predetermined time period, adding a gel precursor ontothe cell layer for gelling, and then further culturing the resultant.Furthermore, Non-patent Document 2 discloses a method that involvesforming a gel layer on a general two-dimensional culture cells and thenperforming a test. Non-patent Document 3 discloses a method thatinvolves previously culturing cells on the surfaces of beads, embeddingthem within a gel matrix, and then performing a test. Moreover,Non-patent Document 4 discloses a method that involves embedding cellspheroids within a gel matrix and then performing a test. Thesethree-dimensional culture methods are attracting attention concerningthe culture of a plurality of cell types; that is, coculture. That isbecause cell behavior more analogous to that observed in vivo can beexpected with the use of coculture compared with culture of a singlecell type.

However, these methods are problematic in that the initial positioncoordinates of single cells or cell aggregates cannot be specified andcontrol of the distribution of cell aggregate sizes is difficult. Hence,application of these methods to an advanced biological test system usinga combination of robotics and viable cell analysis techniques has beenlimited. The use of the method disclosed in Non-patent Document 4basically addresses the above problems since one spheroid is formed ineach well of a formatted well plate. However in this case, a pluralityof spheroids cannot be cultured with determined position coordinatesthereof in each well, so that obtainment of a plurality of data fromeach well and statistical data analysis cannot be performed.Furthermore, a method must be employed to observe spheroids beingembedded in gel under a microscope, which involves harvesting onespheroid prepared per well of a well plate and then dispersing it forcasting in a solution before gelling. Furthermore, the form of a cellaggregate that can be used in the method disclosed in Non-patentDocument 4 is limited to a spheroid.

Meanwhile, in recent years, the field of research using a combination ofa fine processing technology such as semiconductor technology orprinting technology and biotechnology has been significantly developed.There are great expectations for this research, since it may lead todevelopment of new healthcare technology or production of efficient drugdiscovery tools. For example, as disclosed in Non-patent Document 5,Patent Document 1, Patent Document 2, and Patent Document 3, it has beenreported that a culture instrument is produced via application oftechnology for lithographying a photosensitive polymeric material(photoresist) or the like, so as to obtain a two-dimensional cellpattern. Furthermore, Patent Document 4 and Patent Document 5 disclosetechnology that realizes a two-dimensional co-culture system throughcombination with a step of removing a photoresist, so as to use thesystem for a cell function test. Recently, technology for preparing atwo-dimensional cell pattern through application of coating with athickness at the molecular level and the application of such technologyhave been reported. Non-patent Document 6 discloses technology fortesting cell movement with the use of a cell pattern having a cellhaving an artificially controlled adhesion mode as a component.Moreover, Non-patent Document 7 discloses technology for testing cellproliferation activity or the like using an artificial cell patterncontaining cell aggregates as components or using a cell sheet that isformed on an instrument having artificial geometric up and down.Furthermore, Non-patent Document 8 reports a test method that is not anexample of using a cell pattern and involves causing a microgel array onwhich a metabolism enzyme is complexed with a chemical substance to comeinto contact with monolayered culture cells, following which thetoxicity of a metabolite produced by the metabolism enzyme is tested.

Among these examples of technology, a general method using a polymerresist (Non-patent Document 5, Patent Document 1, Patent Document 2, orPatent Document 3) often leads to the production of cell movement testsystems differing from general two-dimensional culture, since the resistgenerally has a degree of thickness that hinders cell movement. Theresulting test systems are often considered to be troublesome artificialtest systems and they have failed to attract attention in applicationfields such as pharmacological tests. In the meantime, the above problemconcerning such resist thickness has been addressed in Patent Document4, Patent Document 5, Non-patent Document 6, and Non-patent Document 7.However, these documents do not mention any biological test method thatinvolves culturing an artificial single cell pattern or a cell aggregatepattern in a matrix. Technology disclosed in Non-patent Document 8 isexpected as a new high-throughput pharmacological test method. However,this is not appropriate as a test method for testing parametersconcerning single cell or cell aggregate movement.

Furthermore, technology that is a combination of fine processingtechnology and three-dimensional culture has been recently reported.Patent Document 6 discloses a method for preparing a spheroid patterncomprising artificially prepared vascular endothelial cells and hepaticcells and the use of the method. Non-patent Document 9 disclosestechnology that involves artificially and three-dimensionally aligningcells in a matrix before gelling and then gelling the matrix. Moreover,Patent Document 7 discloses a method for preparing artificial tissue,which involves transferring a cell layer prepared via pattern culture toa complex composed of a cell layer and a basal membrane layer, so as toprepare artificial tissue. Non-patent Document 10 discloses a methodthat involves pattern culturing cells on collagen gel using a stencilmask, removing the stencil mask, placing a collagen solution on the cellpattern for gelling, and then sandwich culturing the patterned cells.Furthermore, Non-patent Document 11 discloses technology that involvespreparing a polymer pattern through laser ablation, pattern culturingvascular endothelial cells, transferring the cell pattern onto gel,forming gel on the transferred cell pattern, and performingthree-dimensional culture.

However, Patent Document 6 does not disclose any biological test methodthat involves three dimensional culturing of an artificially preparedsingle cell pattern or cell aggregate pattern in a gelled matrix.Non-patent Document 9 discloses technology for culturing a cell patternartificially prepared in gel, but does not disclose any method fortesting a biological indicator concerning the movement of single cellsor cell aggregates prepared in an artificial pattern in a gel matrix.Patent Document 7, Non-patent Document 10, and Non-patent Document 11disclose three-dimensional culture technology for cell aggregatesprepared in an artificial pattern. However, these inventions are notintended for performance of biological tests for parameters concerningthe movement or the proliferation of cell aggregates, and they disclosealmost nothing concerning such parameters. Furthermore, according toPatent Document 7 and Non-patent Document 11, transfer of cells from ahard culture instrument requires approximately 24 hours. This suggeststhat cells strongly interact with such a hard culture instrument. Hence,technology according to Patent Document 7 and Non-patent Document 11 isproblematic in that it is likely to be recognized as a culture systemthat includes artificial factors, compared with conventionalthree-dimensional culture technology. Moreover, the method according toNon-patent Document 10 is problematic in that freedom to design a cellpattern is limited since use of a stencil mask is an essentialrequirement.

Patent Document 1 JP Patent Publication (Kokai) No. 3-7576 A (1991)Patent Document 2 JP Patent Publication (Kokai) No. 5-176753 A (1993)Patent Document 3 JP Patent No. 2777392

Patent Document 4 U.S. Pat. No. 6,133,030Patent Document 5 U.S. Pat. No. 6,221,663

Patent Document 6 WO2003/010302 Patent Document 7 JP Patent Publication(Kokai) No. 2005-342112 A Non-patent Document 1 The FASEB Journal, vol.10, 1471-1484 (1996) Non-patent Document 2 Molecular Biology of theCell, vol. 13, 2474-2485 (2002) Non-patent Document 3 TissueEngineering, vol. 11, no. 1/2, 257-266 (2005) Non-patent Document 4 TheFASEB Journal, vol. 15, 447-457 (2001) Non-patent Document 5 Journal ofBiomedical Materials Research, vol. 32, 165-173 (1996) Non-patentDocument 6 Proceedings of the National Academy of Sciences, vol. 102,no. 4, 975-978 (2005) Non-patent Document 7 Proceedings of the NationalAcademy of Sciences, vol. 102, no. 33, 11594-11599 (2005) Non-patentDocument 8 Proceedings of the National Academy of Sciences, vol. 102,no. 4, 983-987 (2005) Non-patent Document 9 Nature Methods, vol. 3, no.5, 369-375 (2006) Non-patent Document 10 Journal of Biomedical MaterialResearch, vol. 52, 346-353 (2000) Non-patent Document 11 Report ofResearch Support 2000 (ISSN 0916-3719) Japan Cardiovascular ResearchFoundation SUMMARY OF THE INVENTION

The present invention provides a biological test method using culturecells and a test kit for the method, neither of which have beensufficiently achieved by the above known technologies. Specifically, thepresent invention provides a method for performing a biological testunder conditions in which a cell pattern comprising artificiallyprepared single cells or cell aggregates, the initial positioncoordinates of which can be determined, is three-dimensionally culturedwithin a gelled matrix or a condition in which such cell pattern ispseudo-three-dimensionally cultured using a culture instrument thatweakly interacts with cells and a gelled matrix. In particular, thepresent invention provides a method for testing parameters concerningthe movement of a single cell or a cell aggregate. The present inventionfurther provides a test kit that is suitable for realization of suchmethod.

The term “cell aggregate (cell population)” in this specification isdefined as follows. The term “cell aggregate (cell population)” refersto a condition in which a plurality of cells aggregate through somecell-to-cell adhesion at least in the early phase of three-dimensionalculture in a test. In addition, it goes without saying that cell-to-celladhesion may change over the course of a test.

The present invention encompasses the following (1) to (14).

(1) A biological test method, comprising testing a biological indicatorwith reference to at least one selected from the group consisting ofcell proliferation, cell movement, and cell differentiation in a cellpattern substantially embedded in gel.

(2) The biological test method according to (1), wherein the cellpattern is entirely embedded in gel, a portion of the cell pattern isexposed and the remaining portion is embedded in gel, or a portion ofthe cell pattern is in contact with a solid substrate and the remainingportion is embedded in gel.

(3) The biological test method according to (1) or (2), wherein the gelis gel containing at least one type of protein contained in anextracellular matrix, gel containing a pseudo-extracellular matrix, acell sheet, or a complex thereof. (4) The biological test methodaccording to any one of (1) to (3), wherein another cell is present inthe gel.

(5) The biological test method according to any one of (1) to (4),wherein the cell pattern substantially embedded in the gel is formed bya method that involves performing cell culture on a culture instrumenthaving a culture surface on which a cell pattern can be formed and thencoating the culture surface with the gel after culture.(6) The biological test method according to (5), wherein the cellpattern substantially embedded in the gel is formed by a method thatinvolves performing cell culture on a culture instrument having aculture surface on which a cell pattern can be formed, coating theculture surface with the gel after culture, transferring the cellpattern into the gel, and then peeling off the culture instrument.(7) The biological test method according to (6), wherein the cellpattern substantially embedded in the gel is formed by a method thatinvolves performing cell culture on a culture instrument having aculture surface on which a cell pattern can be formed, coating theculture surface with the gel after culture, transferring the cellpattern into the gel, peeling off the culture instrument, and thenfurther coating with gel the face from which the culture instrument onthe gel has been peeled off.(8) The biological test method according to any one of (5) to (7),wherein the culture surface of the culture instrument is provided with acell adhesion region and a cell-adhesion-inhibiting region, the celladhesion region is formed with a film prepared to have cell adhesionproperties by subjecting a cell-adhesion-inhibiting hydrophilic filmcontaining an organic compound having a carbon-oxygen bond to oxidationtreatment and/or degradation treatment, and the cell-adhesion-inhibitingregion is formed with a hydrophilic film containing an organic compoundhaving a carbon-oxygen bond.(9) The biological test method according to any one of (5) to (7),wherein the culture surface of the culture instrument is provided with acell adhesion region and a cell-adhesion-inhibiting region, the celladhesion region and the cell-adhesion-inhibiting region are each formedwith a hydrophilic film containing an organic compound having acarbon-oxygen bond, and the density of the organic compound in the celladhesion region is lower than that of the organic compound in thecell-adhesion-inhibiting region.(10) The biological test method according to any one of (5) to (9),wherein the culture surface of the culture instrument is provided with acell adhesion region and a cell-adhesion-inhibiting region and adifference in height between these regions is 10 nm or less.

(11) A biological test kit, comprising a culture instrument having aculture surface on which a cell pattern can be formed and gel.

(12) The biological test kit according to (11), wherein the culturesurface of the culture instrument is provided with a cell adhesionregion and a cell-adhesion-inhibiting region and the cell adhesionregion is formed with a film prepared to have cell adhesion propertiesby subjecting a cell-adhesion-inhibiting hydrophilic film containing anorganic compound having a carbon-oxygen bond to oxidation treatmentand/or degradation treatment, and the cell-adhesion-inhibiting region isformed with a hydrophilic film containing an organic compound having acarbon-oxygen bond.(13) The biological test kit according to (11), wherein the culturesurface of the culture instrument is provided with a cell adhesionregion and a cell-adhesion-inhibiting region, the cell adhesion regionand the cell-adhesion-inhibiting region are each formed with ahydrophilic film containing an organic compound having a carbon-oxygenbond, and the density of the organic compound in the cell adhesionregion is lower than that of the organic compound in thecell-adhesion-inhibiting region.(14) The biological test kit according to any one of (11) to (13),wherein the culture surface of the culture instrument is provided with acell adhesion region and a cell-adhesion-inhibiting region and adifference in height between these regions is 10 nm or less.

EFFECT OF THE INVENTION

The present invention makes it possible to culture cell aggregateshaving position coordinates that can be determined and having anarbitrary size such that there are almost the same numbers of cells inthe cell aggregates in a gelled matrix or under an environmentequivalent to such a matrix. In particular, the present invention makesit possible to realize biological tests involving high-levelthree-dimensional culture, such as a vascularization test with betterquantitative capability than conventional methods, a vascular remodelingtest that involves simulation of in vivo vascular development orvascularization, a pharmacological test for a compound that inhibits themigration or infiltration of individual cells or cell aggregates, a testconcerning epithelium-stroma transfer (and, in particular,epithelium-stroma transfer of cell aggregates), a test concerning themetastasis of cancer tissue, an artificial matrix performance test usingmodel cells, and the like.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2006-305769, which are priority documents of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross-sectional view of a test system comprising cellpatterns (2), wherein most of each cell pattern is embedded in gel layer(1) and partially exposed.

FIG. 2 shows a cross-sectional view of a test system comprising cellpatterns (2), wherein the cell patterns (2) are arranged between two gellayers (1) and (3) and are entirely embedded in gel.

FIG. 3 shows a cross-sectional view of a test system comprising cellpatterns (2), wherein each cell pattern is partially in a contact withsolid substrate (4) and the remaining portion thereof is embedded in gellayer (1).

FIG. 4 shows a cross-sectional view of a test system comprising cellpatterns (6) and cell patterns (8) that are arranged via lamination,wherein each cell pattern (6) is arranged between two gel layers (5) and(7) and is entirely embedded within gel and each cell pattern (8) isarranged between two gel layers (7) and (9) and is entirely embedded ingel.

FIG. 5 shows a cross-sectional view of a test system, wherein most ofeach cell pattern (11) is embedded in gel layer (10), most of each cellpattern (12) is embedded in gel layer (13), and each cell pattern (11)is arranged to face each cell pattern (12) so that they come intocontact with each other.

FIG. 6 shows a plan view of a test system, wherein a cell pattern (14)and a substance (15) to be tested in terms of effect on the cell patternare positioned and arranged within gel layer (16).

EXPLANATION OF REFERENCE NUMERALS

-   1, 3, 5, 7, 9, 10, 13, and 16 . . . Gel layer-   2, 6, 8, 11, 12, and 14 . . . Cell patterns-   4 . . . Solid substrate (culture instrument)-   15 . . . Substance to be tested-   L . . . Distance between cell pattern (14) and substance to be    tested (15)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (Cell Pattern)

In the present invention, “cell pattern” refers to a pattern formed withsingle cells or cell aggregates that are artificially positioned andarranged. Initial artificial positioning of cells makes it possible tospecify the position coordinates of cells or cell aggregates at thestart of a biological test. Furthermore, initial artificial positioningof cells facilitates microscopic observation and makes it possible toperform statistical data analysis. Moreover, cell patterns can beprepared to have any sizes. Cell aggregates can be prepared so that theyare all composed of almost the same number of cells, while minimizingthe variation in such number. Furthermore, a plurality of cells or cellaggregates can be arranged at any intervals, facilitating observation ofinteraction between a plurality of cells or cell aggregates arranged atintervals according to the relevant test content. Furthermore, theposition coordinates of a cell pattern can be determined. Accordingly,distance L between a substance to be tested (15) and a cell pattern (14)that comprises cells or cell aggregates as shown in FIG. 6 isarbitrarily determined using a commercially available spotter or thelike. Hence, local arrangement of the substance to be tested (15) in gel(16) is also facilitated. Examples of such cell pattern include a cellpattern in which single cells or spheroids (spherical cell aggregates)are arranged at predetermined coordinates, a cell pattern in which aplurality of single cells or spheroids are arranged at predeterminedintervals, and a cell pattern in which cell aggregates are arranged soas to form a predetermined shape such as a line, tree, a network, alattice, a circle, or a quadrangle. A cell pattern formed with cellaggregates may be one-dimensionally-shaped, two-dimensionally-shaped, orthree-dimensionally-shaped. An example of a one-dimensional cell patternis a cell pattern formed with cell aggregates wherein cells are linearlyaligned. An example of a two-dimensional cell pattern is a cell patternformed with planar cell aggregates that are formed with skin epidermalcells, for example. An example of a three-dimensional cell pattern is acell pattern formed with spheroids, tubular cell aggregates that areformed with, vascular endothelial cells, or the like. Furthermore,two-dimensional cell aggregates can be multilayered to compose athree-dimensional cell pattern. In addition, a one-dimensional or atwo-dimensional cell pattern may grow or become deformedthree-dimensionally within gel so as to compose a three-dimensional cellpattern.

Cells composing a cell pattern can be adequately selected according tothe purpose of a test. In particular, mammalian cells having adhesionproperty are useful. Suspended cells such as leukocytes can also be usedin a system where appropriate ligands and cells coexist. Examples ofmammalian species from which cells are derived include mice, rats,monkeys, dogs, pigs, and humans. Furthermore, cells composing a cellpattern may be normal cells represented by primary cells, abnormal(pathological) cells represented by primary cells or cells ofestablished cell lines obtained from the malignant tumor of a patient,or cells of established cell lines that have acquired infiniteproliferation ability in the course of subculture of normal cells. Cellscomposing a cell pattern may be embryonic stem cells, multipotent stemcells, precursor cells having limited differentiation ability, ordifferentiated cells that have completed differentiation. Origins oforgans are not particularly limited. Examples of such cells derived fromorgans include hepatic cells that are hepatic parenchymal cells, Kupffercells, endothelial cells such as vascular endothelial cells and cornealendothelial cells, fibroblasts, osteoblasts, osteoclasts, cells derivedfrom periodontal ligament, epidermal cells such as epidermalkeratinocytes, epithelial cells such as tracheal epithelial cells,gastrointestinal epithelial cells, cervical epithelial cells, cornealepithelial cells, and mammary gland epithelial cells, and pericytes,muscle cells such as smooth muscle cells and cardiac muscle cells, renalcells, islets of Langerhans cells, nerve cells such as peripheralneuronal cells and optic nerve cells, chondrocytes, and bone cells. Inaddition, a single type of cell may be cultured or two or more types ofcell may be co-cultured.

Another test system that maximizes exertion of the characteristics ofthe present invention is employed to conduct a three-dimensional culturetest that uses cell aggregates each consisting of two or more cellshaving cell-to-cell binding as an element of a cell pattern whilemaintaining the cell-to-cell binding as far as possible.

Another test system that is thought to maximize exertion of thecharacteristics of the present invention is employed when an in vitrothree-dimensional culture system is used to test cell aggregates interms of motility or morphological changes involved in cancermetastasis, such as Epithelial-Mesenchymal Transition (EMT).

Another test system that is thought to maximize exertion of thecharacteristics of the present invention is employed when the distancebetween cell aggregates and a substance to be tested in gel or thedistance between cell aggregates and another cell type (interactionbetween them is to be observed) is determined and then athree-dimensional culture test is performed.

(Gel)

Gel to be used in the present invention is not particularly limited, aslong as a cell pattern embedded therein can exert functions such asproliferation, movement, and differentiation. Specific examples of suchgel include artificial hydrogel such as a hydrophilic polymer (e.g.,polyacrylic acid, polyvinyl alcohol, and polyethylene glycol) andhydrogel made of a synthetic substance such as an artificial peptide(e.g., PuraMatrix™). Further examples of the same include gel made ofpolysaccharides such as gel made of hyaluronic acid or a derivativethereof used therein, and gel made of dextran or a derivative thereofused therein. An example of gel made of protein is gel made of varioustypes of collagen, gelatin, fibrin gel, or the like. An example of aspecial extracellular matrix is gel made of a biological site-specificprotein such as a basal membrane represented by Matrigel™. Furthermore,gel made of these complexes can also be used. Among these types of gel,gel containing an organism-derived raw material as an element isdesired. Many biological test case examples concerning such gel havebeen accumulated, so that individual test methods to which the presentinvention is applied can be developed based on the case examples.Cross-linking reaction for gelling may be physical, chemical, orphysical and chemical reaction. Gel made of an artificial peptide isalso preferable. This is because: gel can be produced by totalsynthesis; and it is expected that the quality (e.g., biologicalactivity and gelling ability) of such gel can be easily controlled so asto improve reproducibility in a test. Furthermore, in the presentinvention, a cell sheet that can be said to be living gel is alsoapplicable. When necessary, another cell may also be present in gel anda drug for a test may also be contained.

(Embedding of Cell Pattern in Gel)

The present invention is characterized in that a cell pattern issubstantially embedded in gel. The phrase “substantially embedded ingel” means that a portion of a cell pattern may be exposed or in contactwith a portion other than gel so as not to affect the functions of thecell pattern, such as proliferation, movement, and differentiation.Specifically, the test method of the present invention can be performedunder conditions in which a cell pattern is entirely embedded in gel. Inaddition to such conditions, the test method can also be performed underconditions in which a portion of a cell pattern is exposed in liquidmedium other than gel or air or under conditions in which a cell patternis in contact with a solid substrate, so as not to affect the functionsof the cell pattern. FIGS. 1 to 5 show typical examples of the testsystem of the present invention. FIG. 1 shows a cross-sectional view ofa test system comprising cell patterns (2) wherein most of each cellpattern is embedded in gel layer (1) and partially exposed to theperipheral culture solution or the like. FIG. 2 shows a cross-sectionalview of a test system comprising cell patterns (2), wherein the cellpatterns (2) are arranged two gel layers (1) and (3) and are entirelyembedded in gel. FIG. 3 shows a cross-sectional view of a test systemcomprising cell patterns (2), wherein each cell pattern is partially incontact with solid substrate (4) and the remaining portion thereof isembedded in gel layer (1). FIG. 4 shows a cross-sectional view of a testsystem comprising cell patterns (6) and cell patterns (8) that arearranged via lamination, wherein each cell pattern (6) is arrangedbetween two gel layers (5) and (7) and is entirely embedded in gel andeach cell pattern (8) is arranged between two gel layers (7) and (9) andis entirely embedded in gel. FIG. 5 shows a cross-sectional view of atest system, wherein most of each cell pattern (11) is embedded in gellayer (10), most of each cell pattern (12) is embedded in gel layer(13), and cell pattern (11) is arranged to face cell pattern (12) sothat they come into contact with each other. When a cell pattern isentirely embedded in gel, cells composing the cell pattern can bethree-dimensionally cultured. Moreover, even when a portion of a cellpattern is exposed or in contact with a portion other than gel so as notto affect the functions of cell proliferation, cell movement, celldifferentiation, and the like, substantial three-dimensional culture canbe performed. A culture solution appropriate for cell culture or abuffer appropriate for a biological test may be present inside or in theperiphery of gel.

A cell pattern can be embedded into gel by the following procedures.Cells are seeded on the culture surface of a culture instrument having aculture surface on which a cell pattern can be formed. Cells are thencultured, a cell pattern is formed on the culture surface, and then theculture surface is coated with a gel layer. Hence, as shown in FIG. 3,cell patterns (2) that are partially in contact with solid substrate (4)and the remaining portions are embedded in gel layer (1) are obtained.Furthermore when cell patterns (2) are transferred into gel layer (1)and then culture instrument (solid substrate) (4) is peeled off, cellpatterns (2) with portions exposed as shown in FIG. 1 and with theremaining portions embedded in gel layer (1) are obtained. Furthermore,when the surface of gel layer (1) from which the culture instrument ispeeled off is coated with gel layer (3), cell patterns (2) embedded intwo gel layers (1) and (3) are obtained as shown in FIG. 2. Furthermore,the test systems in FIGS. 4 and 5 can be produced with the use ofsimilar steps. When two or more gel layers are used, gel layers may beof the same type or different types and the interface between the layersmay be clear or unclear.

(Culture Instrument)

A culture instrument to be used for obtaining an artificial cell patternhas a culture surface on which cells are arranged in a predeterminedpattern. Such culture surface is typically provided with cell adhesionregions and cell-adhesion-inhibiting regions, on which cells or cellaggregates are patterned along the shape or the arrangement of such celladhesion regions. Examples of materials for such cell adhesion regionsinclude synthetic polymers such as polystyrene, polyester, and asilicone resin, glass, metals such as gold and titanium, hydrophilicsynthetic polymers to which cell adhesion property is imparted to someextent, such as denatured polyacrylamide, denatured polyacrylic acid,denatured polyethylene glycol, and denatured polyvinyl alcohol. Examplesof materials for such cell-adhesion-inhibiting regions includepolyacrylic acid, polyethylene glycol, Pluronics®, polyacrylamide,polyvinyl alcohol, agar, and albumin. More preferred embodiments of sucha cell adhesion region and cell-adhesion-inhibiting region are asexplained below.

There are two typical embodiments of the culture surface provided withcell adhesion regions and cell-adhesion-inhibiting regions. In a firstembodiment: cell adhesion regions are each formed with a film preparedby subjecting a cell-adhesion-inhibiting hydrophilic film containing anorganic compound having a carbon-oxygen bond to oxidation treatmentand/or degradation treatment, so as to impart cell adhesion propertiesto the resulting film; and cell-adhesion-inhibiting regions are eachformed with a hydrophilic film containing an organic compound having acarbon-oxygen bond. In a second embodiment: cell adhesion regions andcell-adhesion-inhibiting regions are each formed with a hydrophilic filmcontaining an organic compound having a carbon-oxygen bond so thatdensity of the organic compound in the cell adhesion region is lowerthan density of the organic compound in the cell-adhesion-inhibitingregion. Both embodiments are adequate for the present invention butparticularly the first embodiment is preferable.

A particularly preferable embodiment of a culture instrument will bedescribed in detail as follows.

(Substrate of Culture Instrument)

A substrate that composes a culture instrument is not particularlylimited, as long as the substrate is formed with a material so that anorganic compound coating having a carbon-oxygen bond can be formed onthe surface. Specific examples of such material include inorganicmaterials such as metal, glass, ceramic, and silicon and organicmaterials represented by elastomer and plastic (e.g., a polyester resin,a polyethylene resin, a polypropylene resin, an ABS resin, nylon, anacrylic resin, a fluorine resin, a polycarbonate resin, a polyurethaneresin, a methylpentene resin, a phenol resin, a melamine resin, an epoxyresin, and a vinyl chloride resin). Shape of the substrate is also notlimited and examples of the shape include planar shapes such as a flatplate, a flat membrane, a film, and a porous membrane and stereoscopicshapes such as a shape of Petri dish, a cylindrical shape, a shape ofstamp, a shape of a multiwell plate, and a shape of a micro-flow path.When a culture instrument is in the form of a container such as Petridish, culture can be performed using such culture instrument alone. Whena culture instrument is not in the form of a container but in the formof flat plate, for example, such culture instrument may be used incombination with a general culture container. When there is a need toincrease oxygen concentration upon cell culture or when there is a needto homogenize oxygen concentration distribution (concentration gradient)that is obtained by arranging cells (embedded in gel) between an solidsubstrate and an appropriate culture container as in the configurationof FIG. 3, a substrate with high oxygen permeability is preferably used.Examples of materials for forming such substrate include a siliconeresin and silicone hydrogel that is used for high-oxygen-permeabilitysoft contact lenses.

(Cell-Adhesion-Inhibiting Region)

A cell-adhesion-inhibiting region is preferably formed with ahydrophilic film that is formed with an organic compound having acarbon-oxygen bond. Such hydrophilic film is not particularly limited,as long as it is a thin film that is made of, as a main material, anorganic compound having a carbon-oxygen bond and having water-solubilityor water-swelling properties, has cell-adhesion-inhibiting propertiesbefore oxidation, and has cell adhesion properties after oxidationand/or degradation.

The term “carbon-oxygen bond” means a bond that is formed between carbonand oxygen and may not only be a single bond, but also be a double bond.Examples of such carbon-oxygen bond include a C—O bond, a C(═O)—O bond,and a C═O bond.

Examples of such main raw material include water-soluble polymers,water-soluble oligomers, water-soluble organic compounds, surfactants,and amphiphiles. These materials physically or chemically cross-linkeach other to physically or chemically bind to a substrate, therebyforming a hydrophilic thin film.

Specific examples of such a water-soluble polymer material includepolyalkyleneglycol and a derivative thereof, polyacrylic acid and aderivative thereof, polymethacrylic acid and a derivative thereof,polyacrylamide and a derivative thereof, polyvinyl alcohol and aderivative thereof, a zwitterionic polymer, and polysaccharides.Examples of the molecular shapes thereof include linear shapes, branchedshapes, and dendrimers. More specific examples of such materialsinclude, but are not limited to, polyethylene glycol, a copolymer ofpolyethylene glycol and polypropylene glycol (e.g., Pluronic F108,Pluronic F127, poly(N-isopropylacrylamide), poly(N-vinyl-2-pyrrolidone),poly(2-hydroxyethylmethacrylate), andpoly(methacryloyloxyethylphosphorylcholine)), a copolymer ofmethacryloyloxyethylphosphorylcholine and acrylic monomer, dextran, andheparin.

Specific examples of water-soluble oligomer materials and water-solublelow-molecular-weight compounds include an alkylene glycol oligomer and aderivative thereof, an acrylate oligomer and a derivative thereof, amethacrylate oligomer and a derivative thereof, an acrylamide oligomerand a derivative thereof, a saponifiable substance of a vinyl acetateoligomer and a derivative thereof, an oligomer comprising zwitterionicmonomers and a derivative thereof, acrylic acid and a derivativethereof, methacrylic acid and a derivative thereof, acrylamide and aderivative thereof, a zwitterionic compound, a water-soluble silanecoupling agent, and a water-soluble thiol compound. More specificexamples include, but are not limited thereto, an ethylene glycololigomer, an (N-isopropylacrylamide) oligomer, amethacryloyloxyethylphosphorylcholine oligomer, a low-molecular-weightdextran, low-molecular-weight heparin, oligoethylene glycol thiol,ethylene glycol, diethylene glycol, triethylene glycol, tetraethyleneglycol, 2-[methoxy(polyethyleneoxy)-propyltrimethoxysilane, andtriethylene glycol-terminated-thiol.

A hydrophilic film has desirably high cell-adhesion-inhibitingproperties before treatment but exhibits weak cell adhesion propertiesafter oxidation treatment and/or degradation treatment.

The average thickness of such hydrophilic film preferably ranges from0.8 nm to 500 μm, more preferably ranges from 0.8 nm to 100 μm, furthermore preferably ranges from 1 nm to 10 μm, and most preferably rangesfrom 1.5 nm to 1 μm. A hydrophilic film having an average thickness of0.8 nm or more is preferred because such film is hardly affected byregions on the substrate surface not coated with the hydrophilic thinfilms upon protein adsorption or cell adhesion. With a hydrophilic filmhaving an average thickness of 500 μm or less, coating can be relativelyeasily performed.

Examples of a method for forming a hydrophilic film onto a substratesurface include a method that involves causing direct adsorption of ahydrophilic organic compound to a substrate, a method that involvesdirect coating of a substrate with a hydrophilic organic compound, amethod that involves coating a substrate with a hydrophilic organiccompound and then performing cross-linking treatment, a method thatinvolves forming a hydrophilic thin film in a multiple-step manner so asto enhance its adhesiveness to a substrate, a method that involvesforming a foundation layer on a substrate so as to improve adhesivenesswith the substrate and then coating the substrate with a hydrophilicorganic compound, and a method that involves forming a polymerizationinitiation point on a substrate surface and then polymerizinghydrophilic polymer brush.

Examples of particularly preferable methods among the above filmformation methods include a method that involves forming a hydrophilicthin film in a multiple-step manner and a method that involves forming afoundation layer on a substrate so as to improve adhesiveness with thesubstrate and then coating the substrate with a hydrophilic organiccompound. This is because the use of these methods facilitatesenhancement of the adhesiveness of a hydrophilic organic compound to asubstrate. In this specification, the term “binding layer” is used.“Binding layer” means a layer that is present between a hydrophilic thinfilm layer on the outermost surface and a substrate when a hydrophilicorganic compound thin film is formed in a multiple-step manner. When afoundation layer is provided on a substrate surface and a hydrophilicthin film layer is formed on the foundation layer, “binding layer” meansthe foundation layer. Such “binding layer” is preferably a layercontaining a material having a binding portion (linker). Examples of acombination of a linker and a terminal functional group of a material tobe bound to the linker include an epoxy group and a hydroxy group, aphthalic anhydride and a hydroxy group, a carboxyl group and anN-hydroxysuccinimide, a carboxyl group and carbodiimide, and an aminogroup and glutaraldehyde. Either one member of each of thesecombinations may serve as a linker. In these methods, a binding layer isformed with a material having a linker on a substrate before coatingwith a hydrophilic material. Density of the above material in a bindinglayer is an important factor for defining binding strength. Such densitycan be conveniently evaluated using the water contact angle of thesurface of a binding layer as an indicator. For example, in the case ofa silane coupling agent (epoxysilane) having an epoxy group at its end,if the water contact angle of the substrate surface to which epoxysilaneis added is typically 45° or more and desirably 47° or more, a substratehaving sufficient cell-adhesion-inhibiting properties can be prepared bysubsequently adding an ethylene glycol material or the like in thepresence of an acid catalyst.

(Formation of Cell Adhesion Region Via Oxidation Treatment and/orDegradation Treatment of Hydrophilic Film)

In a first embodiment, cell adhesion regions are each formed with a filmprepared by subjecting a cell-adhesion-inhibiting hydrophilic filmcontaining an organic compound having a carbon-oxygen bond to oxidationtreatment and/or degradation treatment, so as to impart cell adhesionproperties to the film. The thus formed cell adhesion regions haverelatively weak cell adhesion ability, so that a pattern of cells orcell aggregates adhering onto the regions can be transferred rapidly toa material that interacts with cells relatively strongly like gel. Suchcell adhesion regions have extremely weak cell adhesion, so thatpolarity of cells quickly changes when the configuration of FIG. 3 isemployed, the cells adhere mainly to a gel layer, and then anenvironment similar to that in the case of three-dimensional culture canbe easily generated. Moreover, even when the configuration of FIG. 3 isemployed, a three-dimensional culture system as configured in FIG. 1 ora three-dimensional culture system as configured in FIG. 2 can beestablished within relatively a short time by peeling off a solidsubstrate (culture instrument) alone.

In the present invention, the term “oxidation” is a narrowly-definedterm and means a reaction by which an organic compound reacts withoxygen so that the content of oxygen is increased compared with thatbefore the reaction.

In the present invention, the term “degradation” indicates a change thatis caused when bonds in the relevant organic compound are cleaved andthen 2 or more types of organic compound are generated from 1 type oforganic compound. Examples of “degradation treatment” include, but arenot limited to, typically degradation by oxidation treatment anddegradation due to ultraviolet irradiation. When “degradation treatment”means degradation accompanying oxidation (that is, oxidativedegradation), “degradation treatment” and “oxidation treatment” indicatethe same treatment.

Degradation due to ultraviolet irradiation indicates degradation thattakes place when an organic compound adsorbs ultraviolet rays and thenis degraded via its excited state. In addition, when a system in whichan organic compound coexists with molecular species containing oxygen(e.g., oxygen and water) is irradiated with ultraviolet rays, inaddition to degradation taking place after adsorption of ultravioletrays by the compound, the molecular species may be activated to reactwith the organic compound. The latter reaction can be classified as“oxidation.” Furthermore the reaction in which an organic compound isdegraded by oxidation by activated molecular species can be classifiednot as “degradation due to ultraviolet irradiation,” but by “degradationdue to oxidation.”

As described above, “oxidation treatment” and “degradation treatment”can overlap operationally and the two are not clearly distinguishable.In this specification, the term “oxidation treatment and/or degradationtreatment” is used.

Examples of an oxidation treatment and/or degradation treatment methodinclude a method that involves treating a hydrophilic film byultraviolet irradiation, a method that involves treating the same byphotocatalytic treatment, and a method that involves treating the samewith an oxidizing agent. A hydrophilic film is partially subjected tooxidation treatment and/or degradation treatment depending on a desiredcell pattern shape. When the film is partially subjected to oxidationtreatment and/or degradation treatment, a mask such as a photomask or astencil mask or a stamp is preferably used. Furthermore, oxidationtreatment and/or degradation treatment may be performed by a directdrawing method such as a method using laser such as ultraviolet laser.

When ultraviolet irradiation treatment is performed, a lamp that ispreferably used as a light source generates ultraviolet rays between theVUV range and the UV-C range. Examples of such lamp include a mercurylamp generating ultraviolet rays with a wavelength of 185 nm or 254 nmand an excimer lamp generating ultraviolet rays with a wavelength of 172nm. When photocatalytic treatment is performed, a light source that ispreferably used herein generates ultraviolet rays with a wavelength of365 nm or less and the same that is more preferably used generatesultraviolet rays with a wavelength of 254 nm or less. As aphotocatalyst, a titanium oxide photocatalyst or a titanium oxidephotocatalyst that is activated using a metal ion or a metal colloid ispreferably used. As an oxidizing agent, organic acid or inorganic acidcan be used without particular limitation. However, since handling ofhigh-concentration acid is difficult, such acid is preferably diluted ata concentration of 10% or less and then used. Optimum time for treatmentwith ultraviolet rays, optimum time for photocatalytic treatment, andoptimum time for treatment with an oxidizing agent can be adequatelydetermined according to various conditions including intensity ofultraviolet rays that are generated from a light source to be used,photocatalyst activity, oxidizing power of an oxidizing agent,concentration of an oxidizing agent, and the like.

(Formation of Cell Adhesion Region Via Achievement of Lower Density ofHydrophilic Film)

In a second embodiment, cell adhesion regions are formed with ahydrophilic film containing a low-density organic compound having acarbon-oxygen bond. The thus formed cell adhesion regions also haverelatively weak cell adhesion ability, so that a pattern of cells orcell aggregates adhering onto the regions can be transferred rapidly toa material that interacts with cells relatively strongly like gel. Suchcell adhesion regions have extremely weak cell adhesion, so thatpolarity of cells quickly changes when the configuration of FIG. 3 isemployed, the cells adhere mainly to a gel layer, and then anenvironment similar to that in the case of three-dimensional culture canbe easily generated. Moreover, even when the configuration of FIG. 3 isemployed, a three-dimensional culture system as configured in FIG. 1 ora three-dimensional culture system as configured in FIG. 2 can beestablished within relatively a short time by peeling off a solidsubstrate (culture instrument) alone.

In this embodiment, both cell adhesion regions andcell-adhesion-inhibiting regions are each formed with a hydrophilic filmcontaining an organic compound having a carbon-oxygen bond. The tworegions differ in terms of the density of the organic compound. There isa tendency that the higher the density, the more hardly the cellsadhere. In the case of the cell adhesion regions, the density of theorganic compound is at a low level such that cells can adhere. On theother hand, in the case of the cell-adhesion-inhibiting regions, thedensity of the organic compound is at a high level such that cellscannot adhere.

An example of a method for controlling the density of such a hydrophilicorganic compound is a method that involves providing a binding layerbetween a hydrophilic organic compound thin film and a substrate surfaceand adjusting strength for binding of the binding layer with thehydrophilic organic compound. Here “binding layer” is as defined aboveand can be composed of preferable materials that are explained above.Regarding the binding strength of a binding layer, the higher thedensity of a material having a linker in the binding layer, the strongerthe binding strength; and the lower the density of the same, the weakerthe binding strength. Density of a material having a linker in a bindinglayer can be conveniently evaluated using the water contact angle of thesurface of a binding layer as an indicator, as described above.

In this embodiment, the density of a material having a linker of abinding layer in cell adhesion regions is low. For example, the watercontact angle of the surface of a binding layer before formation of ahydrophilic organic compound thin film in cell adhesion regionstypically ranges from 10° to 43° and desirably ranges from 15° to 40°when a silane coupling agent (epoxysilane) having an epoxy group at itsend as a material having a linker is used. An example of a method forforming such a binding layer is a method that involves forming a coat(binding layer) made of a material having a linker on a substratesurface and then subjecting the binding layer surface to oxidationtreatment and/or degradation treatment. Examples of a method forsubjecting such a binding layer surface to oxidation treatment and/ordegradation treatment include a method that involves subjecting abinding layer surface to ultraviolet irradiation, a method that involvesperforming photocatalytic treatment, and a method that involvesperforming treatment with an oxidizing agent. A binding layer surfacemay be partially subjected to oxidation treatment and/or degradationtreatment depending on the shape of a desired cell pattern. Partialtreatment can be performed using a mask such as a photomask or a stencilmask or a stamp. Furthermore, oxidation treatment and/or degradationtreatment may also be performed by a direct drawing method such as amethod using laser such as ultraviolet laser. Regarding variousconditions applicable herein, conditions similar to those for a methodfor forming cell adhesion regions through oxidation treatment and/ordegradation treatment performed for a hydrophilic film can be employedherein. Cell adhesion regions can be formed by forming a hydrophilicorganic compound thin film on the thus formed binding layer.

In this embodiment of the present invention, the density of a materialhaving a linker of a binding layer in cell-adhesion-inhibiting regionsis high. For example, the water contact angle of the surface of abinding layer before formation of a hydrophilic organic compound thinfilm in cell-adhesion-inhibiting regions is typically 45° or more anddesirably 47° or more when a silane coupling agent (epoxysilane) havingan epoxy group at its end as a material having a linker is used. Such abinding layer can be obtained by forming a coat made of a materialhaving a linker on a substrate surface. When a binding layer surface ispartially subjected to oxidation treatment and/or degradation treatment,the remaining untreated portion is a binding layer having theabove-mentioned water contact angle. Cell-adhesion-inhibiting regionscan be formed by forming a hydrophilic organic compound thin film on thethus formed binding layer.

(Comparison of Cell Adhesion Region with Cell-Adhesion-InhibitingRegion)

The following explanation is applicable to both embodiments describedabove.

A difference in height between a cell adhesion region (and when abinding layer is present, the binding layer is also included) and acell-adhesion-inhibiting region (and when a binding layer is present,the binding layer is also included) is preferably 10 nm or less. This isbecause such difference in height prevents inhibition of cell movementin a planar direction in the case of a three-dimensional culture withthe configuration of FIG. 3. Either cell adhesion regions orcell-adhesion-inhibiting regions can be convex.

Carbon content in cell adhesion regions (when a binding layer ispresent, the binding layer is also included) is preferably lower thanthat in cell-adhesion-inhibiting regions (when a binding layer ispresent, the binding layer is also included). Specifically, carboncontent in cell adhesion regions preferably ranges from 20% to 99% withrespect to that in cell-adhesion-inhibiting regions. Such carbon contentwithin the range is particularly preferable when hydrophilic filmthickness (the sum of binding layer thickness and hydrophilic filmthickness when a binding layer is present) is 10 μm or less. “Carboncontent (atomic concentration %)” is as defined below.

Moreover, the proportion of carbon (%) binding to oxygen among all thecarbon members in cell adhesion regions (when a binding layer ispresent, the binding layer is also included) is preferably lower thanthe proportion of carbon (%) binding to oxygen among all the carbonmembers in cell-adhesion-inhibiting regions (when a binding layer ispresent, the binding layer is also included). Specifically, theproportion of carbon (%) binding to oxygen among all the carbon membersin cell adhesion regions is preferably 35% to 99% with respect to theproportion of carbon (%) binding to oxygen among all the carbon membersin cell-adhesion-inhibiting regions. Such carbon content within thisrange is particularly preferable when hydrophilic film thickness (thesum of binding layer thickness and hydrophilic film thickness when abinding layer is present) is 10 μm or less. “Proportion of carbonbinding to oxygen (atomic concentration %)” is as defined below.

(Method for Evaluating Hydrophilic Thin Film)

Examples of a technique that can be used for evaluating the hydrophilicthin film (when a binding layer is present, the binding layer is alsoincluded) of the present invention include contact angle measurement,Ellipsometry, observation under an atomic force microscope, observationunder an electron microscope, measurement by Auger electronspectroscopy, measurement by X-ray photoelectron spectroscopy, varioustypes of mass spectroscopy, measurement using a white-lightinterferometer, observation under a confocal laser microscope, andobservation under a probe-type laser microscope. Among these techniques,X-ray photoelectron spectroscopy (XPS/ESCA) has the most excellentquantitative capability. Relative quantitative values can be found bythis measurement technique and are generally calculated based on elementconcentrations (atomic concentrations %). X-ray photoelectronspectroscopy in the present invention is described in detail as follows.

(Method for Calculating Carbon Content in Hydrophilic Thin Film andMethod for Calculating the Proportion of Carbon Binding to Oxygen)

In the present invention, “carbon content” in a hydrophilic thin film isdefined as “carbon content that can be found from the analytical valueof Cls peak obtained using an X-ray photoelectron spectroscopyapparatus.” Furthermore, in the present invention, “proportion of carbonbinding to oxygen” in a hydrophilic thin film is defined as “proportionof carbon binding to oxygen, which is found from the analytical value ofCls peak obtained using an X-ray photoelectron spectroscopy apparatus.”Two specific measurement methods are as described below. In addition,measurement methods in the present invention are not limited to thesemeasurement methods.

(Measurement Method 1)

X-ray photoelectron spectroscopy apparatus: VG_Theta Probe produced byThermo Environmental Instruments Inc.

X-ray source: Monochromated aluminium Kα ray (15 kV−6.67 mA=100 W).

Measurement area: 400 μmφ

Positional relationship between a sample and a detector: A lens fortaking photoelectrons is set at a position having an angle of 53° withrespect to the normal line of the sample

Carbon content: A photoelectron set to be measured is determined throughassumption of elements composing a substrate or a hydrophilic thin film.The concentration of an element (atomic concentration) derived from eachphotoelectron is calculated based on the measured total photoelectronyield that is determined to be 100%. Element concentration (atomicconcentration %) of Cls peak is determined to be a carbon content.

Method for Cls peak fitting: Fitting is performed for C—O bond, C(═O)—Obond, C═O bond, and C—C bond.

Formula for calculating the proportion of carbon binding to oxygen:{[proportion of carbon in C—O bond]+[proportion of carbon in C(═O)—Obond]+[proportion of carbon in C═O bond]}÷{[proportion of carbon in C—Obond]+[proportion of carbon in C(═O)—O bond]+[proportion of carbon inC═O bond]+[proportion of carbon in C—C bond]+[(if necessary) proportionof carbon in the other bonds]}×100(%).

In addition, if necessary, fitting is also performed for the otherbonds. Based on the resulting data, concentration of carbon (atomicconcentration %) in each binding condition of Cls peak is calculated.

(Measurement Method 2)

X-ray photoelectron spectroscopy apparatus: ESCA-3400 (Amicus) producedby KRATOS Analytical.

X-ray source: Non-monochromated magnesium Kα ray (10 kV−20 mA=200 W).

Measurement area: 6 mm φ

Positional relationship between a sample and a detector: A lens fortaking photoelectrons is set on the normal line of the sample

Carbon content: A photoelectron set to be measured is determined throughassumption of elements composing a substrate or a hydrophilic thin film.The concentration of an element (atomic concentration) derived from eachphotoelectron is calculated based on the measured total photoelectronyield that is determined to be 100%. Element concentration (atomicconcentration %) of Cls peak is determined to be a carbon content.

Method for Cls peak fitting: Fitting is performed for C—O bond, C(═O)—Obond, C═O bond, and C—C bond.

Formula for calculating the proportion of carbon binding to oxygen:{[proportion of carbon in C—O bond]+[proportion of carbon in C(═O)—Obond]+[proportion of carbon in C═O bond]}÷{[proportion of carbon in C—Obond]+[proportion of carbon in C(═O)—O bond]+[proportion of carbon inC═O bond]+[proportion of carbon in C—C bond]+[(if necessary) proportionof carbon in the other bonds]}×100(%).

In addition, if necessary, fitting is also performed for the otherbonds. Based on the resulting data, concentration of carbon (atomicconcentration %) in each binding condition of Cls peak is calculated.

(Biological Test Method)

The biological test method of the present invention is to test abiological indicator relating to at least one selected from the groupconsisting of proliferation, movement, and differentiation of cells in acell pattern that is prepared by the above procedures so that the cellpattern is substantially embedded in gel.

Specifically, a biological indicator tested herein relates to at leastone selected from the group consisting of proliferation, movement, anddifferentiation of cells under conditions where gel or medium existingaround a cell pattern contains a drug or cells depending on the purposeof a test. As shown in FIG. 4, a plurality of cells can also be testedsimultaneously in terms of biological indicators with the use of acombination of a plurality of cell patterns. Furthermore, as shown inFIG. 5, cell complexes can be tested in terms of biological indicatorsby causing a plurality of cell patterns to come into contact with eachother. During such a test, cell patterns and gel are preferablymaintained under temperature conditions where cells can undergoproliferation, movement, or differentiation. Gel or medium contains asolvent, a buffer, and the like that are appropriate for cellproliferation, movement, or differentiation if necessary.

Examples of such “a biological indicator relating to at least oneselected from the group consisting of cell proliferation, movement, anddifferentiation” include total amount of genes, cell cycle, number ofcells, ratio of viable cells to dead cells, distance that each cellmoves during an arbitrary time length, direction toward which each cellmoves, variations in cell-to-cell binding, such as tight junction,adherence junction, and gap junction, and enhancement or suppression ofthe expression of various differentiation markers depending on celltypes or differentiation conditions.

According to the present invention, various tests can be performed basedon such biological indicators. Examples of such tests include, but arenot particularly limited thereto, a cytotoxicity test, a chemicalmigration test, a test of a protein phosphorylation inhibitor, a test ofa G protein signal-second messenger inhibitor, a test of a calmodulinphosphorylation protein inhibitor, a test of a cyclin-dependent proteinphosphorylation inhibitor, a test of a MAP phosphorylation-relatedinhibitor, a test of a tyrosine phosphorylation inhibitor, a test of aWnt signal-related inhibitor, a test of an Akt phosphorylation signalinhibitor, a test of a Notch signal inhibitor, a test of a proteindephosphorylation inhibitor, a test of a cytokine signaling inhibitor, atest of a hormone inhibitor, a test of an HDAC inhibitor, a test of anNFκB inhibitor, a test of an agent inhibiting the nucleus-to-cytoplasmtransport of substances, a test of a nervous system-related inhibitor(e.g., calcium signal), a test of a proteinase inhibitor, a test of anagent inhibiting an enzyme that degrades an extracellular matrix, a testof an inhibitor relating to oxidative stress, a test of an apoptosisinducer or inhibitor, a test of a vascularization inducer or inhibitor,a test of a cytoskeleton inhibitor, a test of a cell division inhibitor,a test of a telomerase inhibitor, a test of a saccharificationinhibitor, a test of DNA synthesis inhibitor, and a test of a drug(e.g., antitumor drugs).

A method for culturing cells, which is performed in the biological testmethod of the present invention, is not particularly limited. Forexample, general closed culture using dishes, multiwell plates, or thelike or perfusion culture using bioreactors or the like is employed.

(Observation of Cell Movement)

For observation of cell movement, it is preferable to take movingpictures of cell movement followed by analysis of moving pictures, forexample.

(Method for Taking Moving Pictures)

A typical example of a method for taking moving pictures is as describedbelow. A photographic device that enables observation and recordingunder a microscope at a temperature of 37° C. and with CO₂ concentrationof 5% is used. Appropriate time intervals for taking moving picturesdiffer depending on cells and may be any time intervals as long as thedirection of cell movement does not change significantly. For example,an appropriate time interval ranges from approximately 1 to 5 minutesfor the movement of bovine vascular endothelial cells on glass.

(Method for Analyzing Moving Pictures)

With the use of moving pictures recorded, the cell center of gravity orthe cell nucleus is determined to be the position of the cell and thetime variations of the relevant position coordinate are recorded foreach cell. Examples of quantifiable parameters include velocity vectorsor the population mean thereof, spatiotemporal correlation functions forvelocity, mean-square displacement or a parameter representing its timedependency, and area enclosing cell populations. Furthermore, when cellorientation can be specified such that cells are elliptically shaped, aspatiotemporal correlation function for orientation can also bequantified as a parameter. A velocity vector can be obtained by dividinga displacement vector (obtained when cells migrate at each timeinterval) by the time (required for the migration). When the velocityvectors of a cell population are averaged, how the whole cell populationmoves can be revealed. A spatiotemporal correlation function forvelocity is obtained as the mean cosine of an angle between velocityvectors. A temporal correlation function is obtained via time averagingin terms of specific cells; and a spatial correlation function isobtained via averaging in terms of the certain cell-to-cell distanceusing a fixed time length. A spatiotemporal correlation function is avalue ranging from 0 to 1. The larger the value of a spatiotemporalcorrelation function, the stronger the correlation between velocityvectors and the more uniform movement direction. Mean-squaredisplacement is obtained by averaging the squared values obtained for acell population, each of which is the squared value of the cellmigration distance that cells migrate after a time length (from a timepoint determined to be an initial value). Such mean-square displacementprovides information about how the cell population diffuses. If timedependency of mean-square displacement is supposed to be a valueobtained by raising the time to the power of α, parameter α can beobtained by fitting. When each cell moves randomly, α is 1. “α” closerto 2 means ballistic cell movement. Area S enclosing a cell populationcorresponds to the average value of the squared values each of which isthe squared value of the distance between the cell population center ofgravity and each cell. A spatiotemporal correlation function for cellorientation is obtained by the same calculation method that is employedfor a spatiotemporal correlation function for velocity. Specifically, adirectional vector representing cell orientation is replaced by avelocity vector. The larger the value of a correlation function, thehigher the orientation. It is convenient to use appropriate imageanalysis software for analysis. Examples of free image analysis softwareinclude imageJ (http://rsb.info.nih.gov/ij) and scion image(http://www.scioncorp.com).

(Biological Test Kit)

The present invention further provides a biological test kit containingthe above-mentioned culture instrument and gel. The kit of the presentinvention may also contain a buffer for cell culture, a container (e.g.,multiwell plate) for cell culture, a drug for a test, an instructionmanual, and the like, if necessary. Gel in such kit may also be in theform of solid substances (including powders) that can be gelled by theaddition of a solvent such as water or a buffer when used.

An example of the use of this kit is as follows. Necessary materialssuch as commercial multiwell plates, gel, and test substances areprepared. Some types of cells of specific organs derived from mammals ofdifferent species are pattern-cultured using the above-mentioned cultureinstruments. These cells of mammalian species are cultured species byspecies. The three-dimensional culture test of the present invention isperformed at the same time in different wells of the multiwell plate, sothat differences in terms of action among different species are tested.

EXAMPLES Example 1 Preparation of Substrate for Cell Culture (First-StepReaction)

39.0 g of toluene and 2.25 g of TSL8350 (produced by GE ToshibaSilicone) were mixed. 450 μl of triethylamine was added to the solutionwhile stirring. After several minutes of stirring of the solution atroom temperature, the total volume of the solution was transferred to aglass plate. A 10-cm square glass substrate that had been washed with UVwas immersed in the solution and then the substrate was allowed to standat room temperature for 16 hours. Subsequently, the glass substrate waswashed with ethanol and water and then dried by nitrogen blowing. Thewater contact angle of the substrate surface was approximately 53°.

Second-Step Reaction

25 μl of concentrated sulfuric acid was added dropwise to 50 g oftetraethylene glycol (TEG) while stirring. After several minutes ofstirring of the solution, the total volume of the solution wastransferred to a glass plate. The above substrate was immersed in thesolution, followed by 20 minutes of reaction at 80° C. After reaction,the substrate was washed well with water and then dried by nitrogenblowing. As a result, an organic thin film containing TEG was formed onthe glass substrate surface. The water contact angle of the surface wasapproximately 28°.

(Oxidation Treatment)

A photomask coated with a titanium oxide photocatalyst was prepared. Thephotomask used herein comprises a linear pattern (wherein openings eachhaving a width of 60 μm are provided at a pitch of 300 μm) and linearopenings each having a width of 60 μm and orthogonally crossing theaforementioned linear pattern at intervals of 2.5 cm. The photocatalystlayer of the photomask was caused to come into contact with the abovefilm formation face and then installed in an exposure apparatus, so thatultraviolet irradiation was performed from the photomask side. Lightexposure was performed for 35 seconds using a mercury lamp havingilluminance of 20 mW/cm² at a wavelength of 365 nm, so that thehydrophilic thin film on the substrate surface was partially subjectedto oxidative degradation. This substrate was cut into a size of 25 mm×15mm and then the resultant was used as a cell adhesion substrate.

(Cell Culture)

An autoclave-sterilized substrate was arranged within a culturecontainer and then an appropriate amount of MEM medium containing 5%fetal calf serum was added. 2.0×10⁵ bovine aortic vascular endothelialcells were seeded per substrate. After 48 hours of culture within anincubator (37° C., 5% CO₂), cells adhered only to oxidatively degradedregions and reached confluency on the lines each having a width of 60μm.

(Cell Transfer and Three-Dimensional Culture with the Configuration ofFIG. 2)

A collagen solution was prepared on ice using a collagen gel culture kit(Cellmatrix I-A, Nitta gelatin). 500 μl of the solution was spreadflatly over a 28 mm×33 mm well, followed by 10 minutes of gelling at 37°C. 2 ml of MEM medium containing 5% fetal calf serum was added to thegel. The substrate was caused to sink while upside-down in the medium,so that cells that had adhered to the substrate were caused to come intocontact with collagen gel at the bottom. After 4 hours of culture withinan incubator, the substrate was carefully removed using a pair oftweezers. Almost all the cells had tube-like structures in the collagengel. The medium was removed by suction, 250 μl of the collagen solutionwas applied in layers to the cells, and then gelling was performed at37° C. for 10 minutes. Subsequently, 2 ml of MEM medium containing 5%fetal calf serum, to which proliferation factors (a 10 ng/ml vascularendothelial cell proliferation factor, a 10 ng/ml basic fibroblastproliferation factor, and 50 μg/ml heparin) had been added, was added.The three-dimensional culture with the configuration of FIG. 2 was thenperformed. After 24 hours of culture, the phenomenon of new vesselsgrowing from the existing tube-like structures was observed. This is aphenomenon referred to as vascularization, in which proliferation andmigration take place while maintaining cell-to-cell adhesion. This isalso a representative example of collective cell migration. Thereafter,vascularization consecutively took place. At one week after culture,extremely branched capillary-like networks had formed at high density.In contrast, when suramin (50 μM), a type of vascularization inhibitor,was added to the three-dimensional culture system, the vascularizationthereof was completely inhibited.

Example 2 First-Step Reaction

39.0 g of toluene and 1.20 g of TSL8350 (produced by GE ToshibaSilicone) were mixed. 450 μl of triethylamine was added to the solutionwhile stirring. After several minutes of stirring of the solution atroom temperature, the total volume of the solution was transferred to aglass plate. A 10-cm square glass substrate that had been washed with UVwas immersed in the solution and then the substrate was allowed to standat room temperature for 16 hours. Subsequently, the glass substrate waswashed with ethanol and water and then dried by nitrogen blowing. Thewater contact angle of the substrate surface was approximately 51°.

(Second-Step Reaction)

25 μl of concentrated sulfuric acid was added dropwise to 50 g oftetraethylene glycol (TEG) while stirring. After several minutes ofstirring of the solution, the total volume of the solution wastransferred to a glass plate. The above substrate was immersed in thesolution, followed by 20 minutes of reaction at 80° C. After reaction,the substrate was washed well with water and then dried by nitrogenblowing. As a result, an organic thin film containing TEG was formed onthe glass substrate surface. The water contact angle of the surface wasapproximately 28°.

(Oxidation Treatment)

A photomask coated with a titanium oxide photocatalyst was prepared. Thephotomask used herein comprises a linear pattern (wherein openings eachhaving a width of 60 μm are provided at a pitch of 300 μm) and linearopenings each having a width of 60 μm and orthogonally crossing theaforementioned linear pattern at intervals of 2.5 cm. The photocatalystlayer of the photomask was caused to come into contact with the abovefilm formation face and then installed in an exposure apparatus, so thatultraviolet irradiation was performed from the photomask side. Lightexposure was performed for 35 seconds using a mercury lamp havingilluminance of 20 mW/cm² at a wavelength of 365 nm, so that thehydrophilic thin film on the substrate surface was partially subjectedto oxidative degradation. This substrate was cut into a size of 25 mm×15mm and then the resultant was used as a cell adhesion substrate.

(Cell Culture)

An autoclave-sterilized substrate was arranged within a culturecontainer and then an appropriate amount of MEM medium containing 5%fetal calf serum was added. 2.0×10⁵ bovine aortic vascular endothelialcells were seeded per substrate. After 72 hours of culture, cellsadhered only to oxidatively degraded regions and reached confluency onthe lines each having a width of 60 μm.

(Cell Transfer and Three-Dimensional Culture with the Configuration ofFIG. 3)

200 μl of ice-cooled Growth Factor Reduced (GFR) Matrigel (Becton,Dickinson and Company) was spread over a culture container and thenallowed to stand at room temperature for approximately 1 minute. Thesubstrate was gently placed with its cell adhesion face down onto thematrigel. The resultant was allowed to stand for approximately 5 minuteswhile maintaining this condition. After complete gelling, MEM mediumcontaining 5% fetal calf serum was added and then the substrate wasimmersed in the medium. The culture container was transferred into anincubator (37° C., 5% CO₂) and then cells were cultured while beingsandwiched between a substrate and matrigel. 3 hours later, cellpopulations that had adhered onto the substrate significantly shrinkedto form cylindrical masses. This is a phenomenon that ischaracteristically observed when vascular endothelial cells undergolumen formation in response to differentiation signals coming from theextracellular matrix. When a vascularization inhibitor had beenpreviously added to the three-dimensional culture system, shrinkage ofmultiple cells was inhibited or not inhibited according to the relevantpharmacologic action (Table 1). This means that the effect of a drug onlumen formation can be efficiently tested via observation of cellshrinkage that is observed at the initial stage of differentiation. Thistest method could also be performed for normal human umbilical veinendothelial cells in completely the same manner.

TABLE 1 Various drugs' effects of inhibiting lumen formation in the testmethod Inhibition Inhibitor Action mechanism effect Suramin Inhibitionof the binding of a x proliferation factor with its receptor BTInhibition of cell proliferation Δ and MMP activity Amiloride Inhibitionof u-PA activity x Minocycline Inhibition of MMP3 activity xAnti-VE-cadherin Inhibition of cell-to-cell x antibody adhesion RGDpeptide Inhibition of binding with x fibronectin YIGSR-NH₂ peptideInhibition of binding with Δ laminin PP2 Inhibition of tyrosine kinase ∘Src activity LY294002 Inhibition of PI3 kinase Δ activity Akt inhibitorInhibition of Akt kinase x activity Y27632 Inhibition of Rho kinase xactivity ∘ Large inhibition effect, Δ Small inhibition effect, and x Noinhibition effect

Example 3 First-Step Reaction

39.0 g of toluene and 0.56 g of TSL8350 (produced by GE ToshibaSilicone) were mixed. 450 μl of triethylamine was added to the solutionwhile stirring. After several minutes of stirring of the solution atroom temperature, the total volume of the solution was transferred to aglass plate. A 10-cm square glass substrate that had been washed with UVwas immersed in the solution and then the substrate was allowed to standat room temperature for 16 hours. Subsequently, the glass substrate waswashed with ethanol and water and then dried by nitrogen blowing. Thewater contact angle of the substrate surface was approximately 50°.

(Second-Step Reaction)

25 μl of concentrated sulfuric acid was added dropwise to 50 g of TEGwhile stirring. After several minutes of stirring of the solution, thetotal volume of the solution was transferred to a glass plate. The abovesubstrate was immersed in the solution, followed by 2 hours of reactionat 80° C. After reaction, the substrate was washed well with water andthen dried by nitrogen blowing. As a result, an organic thin filmcontaining TEG was formed on the glass substrate surface. The watercontact angle of the substrate surface was approximately 27°.

(Oxidation Treatment)

A photomask coated with a titanium oxide photocatalyst was prepared. Thephotomask used herein comprises a linear pattern (wherein openings eachhaving a width of 60 μm are provided at a pitch of 300 μm) and linearopenings each having a width of 60 μm and orthogonally crossing theaforementioned linear pattern at intervals of 2.5 cm. The photocatalystlayer of the photomask was caused to come into contact with the abovefilm formation face and then installed in an exposure apparatus, so thatultraviolet irradiation was performed from the photomask side. Lightexposure was performed for 35 seconds using a mercury lamp havingilluminance of 20 mW/cm² at a wavelength of 365 nm, so that thehydrophilic thin film on the substrate surface was partially subjectedto oxidative degradation. This substrate was cut into a size of 25 mm×15mm and then the resultant was used as a cell adhesion substrate.

(Cell Culture)

An autoclave-sterilized substrate was arranged within a culturecontainer and then an appropriate amount of MEM medium containing 5%fetal calf serum was added. 2.0×10⁵ bovine aortic vascular endothelialcells were seeded per substrate. After 72 hours of culture, cellsadhered only to oxidatively degraded regions and reached confluency onthe lines each having a width of 60 μm.

(Cell Transfer and Three-Dimensional Culture with the Configuration ofFIG. 3)

200 μl of ice-cooled Growth Factor Reduced (GFR) Matrigel (Becton,Dickinson and Company) was spread over a culture container and thenallowed to stand at room temperature for approximately 1 minute. Thesubstrate was gently placed with its cell adhesion face down onto thematrigel. The resultant was allowed to stand for approximately 5 minutesfor gelling while maintaining this condition. MEM medium containing 5%fetal calf serum was added and then the substrate was immersed in themedium. The culture container was transferred into an incubator (37° C.,5% CO₂) and then cells were cultured while being sandwiched between asubstrate and matrigel. 3 hours later, cell populations that had adheredonto the substrate significantly shrunk to form cylindrical masses whenthey were observed under a phase-contrast microscope. Based on theextremely unclear interface between cells at this time, it was concludedthat cell-to-cell adhesion had been highly developed via lamination ofmatrigel. However, when culture was continued under the same conditions,cell populations at the initial stage of differentiation underwentdedifferentiation within 24 hours and disintegration of cell-to-celladhesion and single cell migration were observed. This three-dimensionalculture system was thought to be a culture system useful for testingswitching between differentiation and dedifferentiation of vascularendothelial cells and for testing the development and disintegration ofcell-to-cell adhesion.

Example 4 Preparation of Substrate for Cell Culture (First-StepReaction)

A solution was prepared by mixing 39 g of toluene, 13.5 g of epoxysilane(TSL8350, GE Toshiba Silicone), and 450 μl of triethylamine. Apredetermined amount of the thus prepared solution was transferred intoa glass Petri dish. A 10-cm square glass plate having a thickness of 0.7mm, which had been washed with UV, was immersed in the solution and thenthe plate was allowed to stand at room temperature for 16 hours forreaction. Subsequently, the glass plate was washed with ethanol,subjected to ultrasonic washing with water, and then dried. The watercontact angle of the glass plate surface was approximately 50.1°.

(Second-Step Reaction)

25 μl of concentrated sulfuric acid was added to 50 g of tetraethyleneglycol while stirring. A predetermined amount of the solution wastransferred to a glass Petri dish. The above epoxidized glass plate wasimmersed in the solution, followed by 20 minutes of reaction at 80° C.After reaction, the substrate was washed with water and then dried. Thesurface water contact angle was measured and the water contact angle was29.4° on an average.

(Oxidation Treatment)

A photomask coated with a titanium oxide photocatalyst was prepared. Thephotomask used herein comprises a linear pattern (wherein openings eachhaving a width of 60 μm are provided at a pitch of 300 μm), linearopenings each having a width of 60 μm and orthogonally crossing theaforementioned linear pattern at intervals of 2.5 cm, and openings eachhaving a width of approximately 1.5 cm provided in the periphery of thephotomask. The illuminance of an exposure apparatus was measured inadvance at a wavelength of 350 nm and the measured value was used as anindicator for determination of time of exposure. The illuminance was18.6 mW/cm². The glass substrate on which a hydrophilic thin film hadbeen formed and the photomask were arranged so that the hydrophilic thinfilm faced the catalyst layer of the photomask and they were installedin the exposure apparatus so that light came from the photomask side.Light exposure was performed for 57 seconds, so that oxidativedegradation was performed. This substrate was then cut into a size of 24mm×15 mm to facilitate the use of the substrate for culture.

(Cell Culture)

Each of the above cut substrate was subjected to high pressure vaporsterilization using an autoclave. The substrate was arranged within aculture container. 1.5×10⁵ vascular endothelial cells (BAEC: bovineaortic endothelial cells) of bovine carotid artery were seeded persubstrate. MEM medium containing 5% serum was used and then cells werecultured for 48 hours within an incubator at 37° C. and with 5% CO₂concentration. When observed under a fluorescence phase-contrastmicroscope, BAECs adhered only to portions subjected to oxidationtreatment.

(Cell Transfer and Three-Dimensional Culture)

200 μl of matrigel (trademark) was spread over a culture container andthen allowed to stand at room temperature for several minutes so thatgelling was performed to some extent. 5×10⁵ cells/mL mouse-derivedfibroblasts that had been fluorescence-labeled with PKH26 in advancewere injected into the Matrigel™. The cell culture substrate was placedso as to face the Matrigel™. Subsequently, D-MEM medium containing 10%serum was added, followed by 3 hours of culture at 37° C. and 5% CO₂within an incubator. At this time point, it was confirmed under afluorescence phase-contrast microscope that BAECs had undergone amorphological change so as to form a tube shape. It was also confirmedunder a fluorescence phase-contrast microscope that almost nofibroblasts had migrated within the matrigel. The substrate wascarefully peeled off using a pair of tweezers. BAECs were transferredonto the Matrigel™ and then cultured for 2 hours at 37° C. and 5% CO₂within an incubator. At this time point, it was confirmed under afluorescence phase-contrast microscope that almost no BAECs and almostno fibroblasts had migrated.

Example 5 Preparation of Substrate for Cell Culture (First-StepReaction)

A solution was prepared by mixing 39 g of toluene, 0.7 g of epoxysilane(TSL8350, GE Toshiba Silicone), and 400 μl of triethylamine. Apredetermined amount of the thus prepared solution was transferred intoa container. A 10-cm square glass plate having a thickness of 0.7 mm,which had been washed with ultraviolet rays, was immersed in thesolution. After 18 hours of reaction at room temperature, the glassplate was washed with toluene, washed with ethanol, and then finallysubjected to ultrasonic washing with water. The water contact angle ofthe glass plate surface was 51.3° on an average.

(Second-Step Reaction)

25 μl of concentrated sulfuric acid was added to 50 g of tetraethyleneglycol while stirring. A predetermined amount of the solution wastransferred to a container. The above epoxidized glass plate wasimmersed in the solution, followed by 20 minutes of reaction at 80° C.After reaction, the substrate was washed well with water. The watercontact angle of the glass plate surface was 32.0° on an average. Thus,the glass plate on which a hydrophilic thin film had been formed couldbe prepared.

(Oxidation Treatment)

A photomask coated with a titanium oxide photocatalyst was prepared. Thephotomask used herein comprises 100 μm-square opening array regionsformed at a pitch of 200 μm, 200 μm-square opening array regions formedat a pitch of 400 μm, 300 μm-square opening array regions formed at apitch of 600 μm, 400 μm-square opening array regions formed at a pitchof 800 μm, and openings each having a width of approximately 1.5 cmformed in the periphery of the photomask. The illuminance of an exposureapparatus was measured in advance at a wavelength of 350 nm and themeasured value was used as an indicator for determination of time ofexposure. The illuminance was 18.6 mW/cm². The glass plate on which ahydrophilic thin film had been formed and the photomask were arranged sothat the hydrophilic thin film faced the photocatalyst layer of thephotomask and they were installed in the exposure apparatus so thatirradiation with ultraviolet rays was performed from the back side of aquartz plate. Light exposure was performed for 161 seconds, so thatoxidative treatment was performed. Subsequently, this substrate was cutinto a size of 24 mm×15 mm for use in culture. Portions facing theopenings in the periphery of the photomask were used for X-rayPhotoelectron Spectroscopy (XPS).

(Surface Analysis)

Hydrophilic thin films were measured before and after oxidationtreatment using a VG_Theta Probe produced by Thermo EnvironmentalInstruments Inc. Cls peak fitting was performed for carbons of C—Cbonds, carbons of C—O bonds, carbons of C(═O)—O bonds and C═O bonds. Thethus obtained carbon content of the hydrophilic thin film measured afteroxidation treatment was 85.3% of the carbon content of the hydrophilicthin film measured before oxidation treatment. Furthermore, theproportion of carbon binding to oxygen in the hydrophilic thin filmmeasured before oxidation treatment was 76.7%; and the proportion ofcarbon binding to oxygen in the hydrophilic thin film measured afteroxidation treatment was 64.6%. Furthermore, the water contact angle ofthe hydrophilic thin film surface measured after oxidation treatment was28.8°. Furthermore, the patterning surface was observed using a scanningwhite-light interferometer (Zygo NewView 5000). An approximately 1-nmdifference in height was formed depending on the mask pattern.

(Cell Culture)

Each of the above cut substrate for pattern culture (on which 200μm-square cell adhesion regions had been formed at a pitch of 400 μm)was subjected to high pressure vapor sterilization using an autoclave.The substrate was arranged within a culture container. 2×10⁵ normalhuman (adult) dermal fibroblasts (KF-4109, Cascade Biologics) dispersedin a culture solution (basal medium M-106-500S, additive agent KE-6350,Cascade Biologics/KURABO) were seeded per substrate. Fibroblasts werecultured for 46 hours under conditions of 37° C. and 5% CO₂ using anincubator. Cell patch arrays comprising monolayered fibroblasts wereformed. Whereas fibroblasts in the patch central portions becamerelatively smaller in size, fibroblasts in the patch peripheral portionswere relatively spindle-shaped.

(Cell Transfer and Three-Dimensional Culture with the Configuration ofFIG. 2)

A reagent was prepared according to the manufacturer's protocols using acollagen gel kit (Cellmatrix I-A, Nitta gelatin). 250 μl of the reagentwas developed over each of the cell culture substrates on a culturecontainer and then the substrates were maintained at room temperaturefor 10 minutes for gelling to some extent. The cell culture substratewas placed on the collagen gel, so that the cell patch arrays faced thecollagen gel. Under the condition, the substrate was allowed to standwithin an incubator at 37° C. for 3 minutes. The above medium was addedand then cells were cultured for 4 hours within the incubator. At thistime point, almost no cells of the cell array patches migrated.Subsequently, the substrate was carefully peeled off using a pair oftweezers and then the cell patch arrays were transferred onto thecollagen gel. The medium was then removed by suction. 250 μl of thecollagen reagent was added to each substrate. The substrate was allowedto stand within the incubator for 30 minutes for gelling of collagen.Subsequently, medium was added and then three-dimensional culture wasperformed with the configuration of FIG. 2. When observed under aphase-contrast microscope, cells in the periphery of the cell patchesinitiated migration after 4 hours of culture. 14 hours later, the cellsfurther migrated but cells in the patch central portions migrated onlyslightly. 40 hours later, cells further migrated and proliferated, sothat cell patches seemed to be connected with each other. Moreover, thecells in the patch central portions became almost spindle-shaped unlikethe original shape at the initiation of three-dimensional culture.Furthermore, 10 μM dimethylsulfoxide (DMSO) was added and then similarthree-dimensional culture was performed and an experiment was conductedwithout using DMSO. When the results of the two experiments werecompared, there were no significant differences in terms of change overtime in cell motility. These results demonstrated that the effects ofvarious inhibitors can be tested using these experiments as controlexperiments.

Example 6 Cell Culture

A substrate for pattern culture used herein was prepared by forming300-μm square cell adhesion regions prepared in Example 5 at a pitch of600 μm. Cells and medium same as those used in Example 5 were used.4×10⁵ normal human (adult) dermal fibroblasts (NHDF) were seeded persubstrate, an appropriate amount of medium was added, and then cellswere cultured within an incubator. 23 hours later, when observed under aphase-contrast microscope, cell spheroid arrays were formed.Furthermore, 1.8X10⁵ NHDFs were seeded per substrate, so that cell patcharrays were prepared.

(Embedding of Cell Aggregate into Gel)

The above cell spheroid arrays and cell patch arrays were embedded intogel that had been prepared from the collagen gel kit used in Example 5.Three-dimensional culture was performed with the configuration ofFIG. 1. Cell movement and cell proliferation were observed over time. Asa result of this experiment, in the case of the cell spheroid arrays,the cells in the regions that had initially adhered to the patternculture substrate were the first cells that began their migration. Interms of cell migration distance per unit time, there were nosignificant differences between the cells in the regions that hadinitially adhered to the pattern culture substrate and the cells in theperiphery of the patches of the cell patch arrays. On the other hand,spheroids' own migration was not observed for at least approximately 15hours after the initiation of culture. At 40 hours after the initiationof culture, the density of spheroids' main bodies became lower and cellinfiltration from the spheroids' main bodies was observed.

Example 7 Substrate Preparation for Cell Culture (First-Step Reaction)

A solution was prepared by mixing 39 g of toluene, 13.5 g of epoxysilane(TSL8350, GE Toshiba Silicone), and 450 μl of triethylamine. Apredetermined amount of the thus prepared solution was transferred intoa glass Petri dish. A glass plate having a diameter of 31 mm and athickness of approximately 0.1 mm, which had been washed withultraviolet rays, was immersed in the solution, followed by 18 hours ofreaction at room temperature. The glass plate was washed with ethanol,subjected to ultrasonic washing with water, and then dried. The watercontact angle of the glass plate surface was 51.1° on an average.

(Second-Step Reaction)

25 μl of concentrated sulfuric acid was added to 50 g of tetraethyleneglycol while stirring. A predetermined amount of the solution wastransferred to a glass Petri dish. The above epoxidized glass plate wasimmersed in the solution, followed by 20 minutes of reaction at 80° C.After reaction, the substrate was washed with water and then dried. Thewater contact angle of the surface was measured. The water contact anglewas 30.2° on an average.

(Oxidation Treatment and Preparation of Patterning Bottom Dish)

Patterning glass was obtained by 180 seconds of exposure under theconditions same as those in Example 4 and oxidation treatment followingthereto. The glass was attached onto the underside of a 35-mmpolystyrene dish with a hole having a diameter of 27 mm, so that thehole was covered with the glass.

(Cell Culture)

The above dish was sterilized with 70% ethanol. Normal human (adult)dermal fibroblasts (KF-4109, Cascade Biologics) dispersed in a culturesolution (basal medium M-106-500S, additive agent KE-6350, and CascadeBiologics/KURABO) was seeded at 4×10⁵ cells per dish. Cells werecultured using an incubator under conditions of 37° C. and 5% CO₂ for 18hours. A linear pattern formed by cells that had adhered was obtained.

(Embedding of Cell Aggregate into Gel)

Cell aggregates were embedded into gel that had been prepared using thecollagen gel kit used in Example 5 and then three-dimensional culturewas performed with the configuration of FIG. 3. After 30 minutes ofgelling at 37° C., three types of culture were performed using (1) asystem containing the above culture solution, (2) a system containing aculture solution prepared by adding 10 μM inhibitor PP2 to the aboveculture solution, and (3) a system containing a culture solutioncontaining DMSO with the same concentration as that of DMSO (used as asolvent for preparing 1 mM stock solution of PP2) contained in theabove-prepared culture solution containing PP2. Cell migration and cellproliferation conditions were observed at 3 hours, 6 hours, 9 hours, and21 hours after the initiation of culture. As a result, cell migrationand cell proliferation were significantly suppressed in the system (2)containing the inhibitor, compared with the systems (1) and (3).

Example 8 Cell Culture

Substrates for pattern culture (200 μm-square cell adhesion regions wereformed at a pitch of 400 μm) that had been used in Example 5 and cut inadvance were subjected to high-pressure vapor sterilization using anautoclave. These substrates were arranged in culture containers. BAECwas pattern-cultured using MEM medium containing 5% fetal calf serum.

The following three-dimensional culture was performed using thesubstrate on which pattern culture had been performed for 18 hours andthe substrate on which pattern culture had been performed for 42 hoursand cell-to-cell adhesion had been further developed.

(Embedding of Cell Aggregate into Gel)

With the use of Growth Factor Reduced (GFR) Matrigel (Becton, Dickinsonand Company), three-dimensional culture was performed with theconfiguration of FIG. 3 similarly to Example 2. Time lapse observationwas performed. As a result, there were no significant differencesbetween the system involving 18 hours of pattern culture and the systeminvolving 42 hours of pattern culture in terms of the random nature ofcell movement and the average cell movement velocity. In contrast, therewas a significant difference between the two in terms of increasingamount (of area S enclosing the cell population) per unit time.Specifically, the increasing amount (of area S in the system involving42 hours of pattern culture) per unit time was 1/5 with respect to thatof the system involving 18 hours of pattern culture. This may be due tocompositive effects of cell-to-cell adhesion strength of vascularendothelial cells, shrinkage force of cell aggregates, anddifferentiation and/or dedifferentiation of vascular endothelial cells.

1. A biological test method, comprising testing a biological indicatorwith reference to at least one selected from the group consisting ofcell proliferation, cell movement, and cell differentiation in a cellpattern substantially embedded in gel.
 2. The biological test methodaccording to claim 1, wherein the cell pattern is entirely embedded ingel, a portion of the cell pattern is exposed and the remaining portionis embedded in gel, or a portion of the cell pattern is in contact witha solid substrate and the remaining portion is embedded in gel.
 3. Thebiological test method according to claim 1, wherein the gel is gelcontaining at least one type of protein contained in an extracellularmatrix, gel containing a pseudo-extracellular matrix, a cell sheet, or acomplex thereof.
 4. The biological test method according to claim 1,wherein another cell is present in the gel.
 5. The biological testmethod according to claim 1, wherein the cell pattern substantiallyembedded in the gel is formed by a method that involves performing cellculture on a culture instrument having a culture surface on which a cellpattern can be formed and then coating the culture surface with the gelafter culture.
 6. The biological test method according to claim 5,wherein the cell pattern substantially embedded in the gel is formed bya method that involves performing cell culture on a culture instrumenthaving a culture surface on which a cell pattern can be formed, coatingthe culture surface with the gel after culture, transferring the cellpattern into the gel, and then peeling off the culture instrument. 7.The biological test method according to claim 6, wherein the cellpattern substantially embedded in the gel is formed by a method thatinvolves performing cell culture on a culture instrument having aculture surface on which a cell pattern can be formed, coating theculture surface with the gel after culture, transferring the cellpattern into the gel, peeling off the culture instrument, and thenfurther coating with gel the face from which the culture instrument onthe gel has been peeled off.
 8. The biological test method according toclaim 5, wherein the culture surface of the culture instrument isprovided with a cell adhesion region and a cell-adhesion-inhibitingregion, the cell adhesion region is formed with a film prepared to havecell adhesion properties by subjecting a cell-adhesion-inhibitinghydrophilic film containing an organic compound having a carbon-oxygenbond to oxidation treatment and/or degradation treatment, and thecell-adhesion-inhibiting region is formed with a hydrophilic filmcontaining an organic compound having a carbon-oxygen bond.
 9. Thebiological test method according to claim 5, wherein the culture surfaceof the culture instrument is provided with a cell adhesion region and acell-adhesion-inhibiting region, the cell adhesion region and thecell-adhesion-inhibiting region are each formed with a hydrophilic filmcontaining an organic compound having a carbon-oxygen bond, and thedensity of the organic compound in the cell adhesion region is lowerthan that of the organic compound in the cell-adhesion-inhibitingregion.
 10. The biological test method according to claim 5, wherein theculture surface of the culture instrument is provided with a celladhesion region and a cell-adhesion-inhibiting region and a differencein height between these regions is 10 nm or less.
 11. A biological testkit, comprising a culture instrument having a culture surface on which acell pattern can be formed and gel.
 12. The biological test kitaccording to claim 11, wherein the culture surface of the cultureinstrument is provided with a cell adhesion region and acell-adhesion-inhibiting region and the cell adhesion region is formedwith a film prepared to have cell adhesion properties by subjecting acell-adhesion-inhibiting hydrophilic film containing an organic compoundhaving a carbon-oxygen bond to oxidation treatment and/or degradationtreatment, and the cell-adhesion-inhibiting region is formed with ahydrophilic film containing an organic compound having a carbon-oxygenbond.
 13. The biological test kit according to claim 11, wherein theculture surface of the culture instrument is provided with a celladhesion region and a cell-adhesion-inhibiting region, the cell adhesionregion and the cell-adhesion-inhibiting region are each formed with ahydrophilic film containing an organic compound having a carbon-oxygenbond, and the density of the organic compound in the cell adhesionregion is lower than that of the organic compound in thecell-adhesion-inhibiting region.
 14. The biological test kit accordingto claim 11, wherein the culture surface of the culture instrument isprovided with a cell adhesion region and a cell-adhesion-inhibitingregion and a difference in height between these regions is 10 nm orless.